Mutations play a critical role in the etiology of many diseases, such as autism, schizophrenia, diabetes, and cancer. Most mutations arise during the replication of damaged DNA, a process called translesion synthesis (TLS). TLS is carried out by specialized TLS polymerases, which have evolved to accommodate DNA damage. Several fundamental, unanswered questions about TLS polymerases remain. In Aim 1, we will address the following question: how are TLS polymerases recruited to stalled replication forks? To do this, we will use both ensemble and single-molecule binding assays and steady state kinetics. These studies will reveal the mechanisms of TLS polymerase recruitment to ubiquitin-modified PCNA (UbPCNA), a key component of stalled replication forks. These studies will also reveal the structural motifs required for these interactions. In Aim 2, we will address the following question: how are TLS polymerases selected for recruitment to specific DNA lesions? To do this, we will use ensemble and single-molecule binding assay. These studies will reveal the mechanisms of TLS polymerase selection and the influence of the DNA lesion on this process. In Aim 3, we will address the following question: how are TLS polymerases structurally organized at stalled replication fork? To do this, we will use X-ray crystallography, computational modeling and simulations, and a variety of experimental distance measurements. These studies will provide the first glimpse of the structural organization of TLS polymerases within these protein-DNA complexes and will reveal how the different TLS polymerases are coordinated during the multi-step process of TLS.